The ultimate objective of the proposed research program is to delineate the molecular basis for gene expression during animal growth and development. The focus of the investigation is to purify and characterize the (3DP-Mannose-requiring mannosyltransferases of the dolichol cycle in the rat mammary gland and develop molecular reagents, viz, monospecific polyclonal antibodies against the purified enzymes and cDNAs for the same. These investigations are being proposed in the context of earlier studies from the P.I.'s laboratory showing that several ER-localized enzymes for protein N-glycosylation are modulated during the reproductive cycle for the growth and differentiation of the mammary gland until they reach peak levels at mid-lactation. The combined action of insulin, prolactin, and hydrocortisone appears to regulate their expression above the basal level. A working hypothesis has been formulated according to which the dolichol- linked assembly and the polypeptide-linked maturation of the oligosaccharide precursor for N-glycosylation are coordinately upregulated by the synergistic action of insulin, prolactin and hydrocortisone during the lactogenic differentiation of the mammary gland. Asparagine-linked glycoproteins comprise the largest class of glycoproteins and are involved in a myriad of phenomena that are fundamental to biological recognition. Alterations in glycoprotein metabolism are associated with a variety of pathologies, e.g., malignancy, atherosclerosis, numerous genetic disorders and the initial host-parasite interaction leading to AIDS. A concert of seventeen glycosyltransferases and two glycosidases, viz., glucosidase I and II is minimally required for the assembly of all N-linked glycoproteins. Among these, five GDP-Man- requiring mannosyltransferases appear to constitute a family of enzymes. Because of their membrane-bound characteristic, extreme instability and the lack of well-defined acceptor substrates, these enzymes have consistently defied the efforts of many laboratories to purify them. In the proposed investigation, these enzymes will be purified and characterized, employing novel photoidentification and active site -SH tagging methodologies developed in the P.I.'s laboratory. A specific goal will be to identify a potential "mannosyl motif" within the active site of the enzymes. An experimental strategy is outlined to obtain polyclonal antibodies against individual enzymes even if they resist purification to homogeneity. Standard and PCR-based technologies will be employed to obtain cDNA probes for the mannosyltransferases. The availability of these reagents should open the door for future investigations on the regulation of protein N-glycosylation in the mammary gland and other tissues. The mammary gland offers a unique model to the investigator for studying glycoprotein biosynthesis at the biochemical and molecular-biological level. It is intensely modulated by a variety of hormones for its growth and differentiation throughout the reproductive life of the female. It can potentially serve as an excellent bioreactor in transgenic animals to harvest large quantities of biomedically significant glycoproteins in its secretion, i.e., milk. Preliminary successes with the secretion of alpha- antitrypsin, tissue plasminogen activator, and blood clotting factors appear very promising.